Examination of the roles of a conserved motif in the PriA helicase in structure-specific DNA unwinding and processivity

DNA replication complexes (replisomes) frequently encounter barriers that can eject them prematurely from the genome. To avoid the lethality of incomplete DNA replication that arises from these events, bacteria have evolved “DNA replication restart” mechanisms to reload replisomes onto abandoned replication forks. The Escherichia coli PriA DNA helicase orchestrates this process by recognizing and remodeling replication forks and recruiting additional proteins that help to drive replisome reloading. We have identified a conserved sequence motif within a linker region of PriA that docks into a groove on the exterior of the PriA helicase domain. Alterations to the motif reduce the apparent processivity and attenuate structure-specific helicase activity in PriA, implicating the motif as a potential autoregulatory element in replication fork processing. The study also suggests that multiple PriA molecules may function in tandem to enhance DNA unwinding processivity, highlighting an unexpected similarity between PriA and other DNA helicases.

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DNA Helicase-SSB Interactions Critical to the Regression and Restart of Stalled DNA Replication Forks in Escherichia coli

Genes ◽

10.3390/genes11050471 ◽

2020 ◽

Vol 11 (5) ◽

pp. 471 ◽

Cited By ~ 1

Author(s):

Piero R. Bianco

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Dna Helicase ◽

Dependent Manner ◽

Replication Forks ◽

Replication Restart ◽

Helicase Dnab ◽

Fork Regression ◽

Ob Fold ◽

Dna Replication Restart

In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

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p53 orchestrates DNA replication restart homeostasis by suppressing mutagenic RAD52 and POLθ pathways

eLife ◽

10.7554/elife.31723 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 34

Author(s):

Sunetra Roy ◽

Karl-Heinz Tomaszowski ◽

Jessica W Luzwick ◽

Soyoung Park ◽

Jun Li ◽

...

Keyword(s):

Dna Replication ◽

Genomic Instability ◽

Genome Instability ◽

Single Cells ◽

Replication Forks ◽

Mutational Signatures ◽

Development Of Resistance ◽

Cycle Arrest ◽

Replication Restart ◽

Dna Replication Restart

Classically, p53 tumor suppressor acts in transcription, apoptosis, and cell cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks that drive genomic instability, which isgenetically separable from transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POLθ pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53-mediated suppression of replication genome instability.

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Synthetic DNA Replication Bubbles Bound and Unwound with Twofold Symmetry by a Simian Virus 40 T-Antigen Double Hexamer

Journal of Virology ◽

10.1128/jvi.72.11.8676-8681.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8676-8681 ◽

Cited By ~ 21

Author(s):

Natalia V. Smelkova ◽

James A. Borowiec

Keyword(s):

Dna Replication ◽

Simian Virus 40 ◽

Simian Virus ◽

Dna Helicase ◽

T Antigen ◽

Dna Unwinding ◽

Replication Forks ◽

Helicase Activity ◽

Synthetic Dna ◽

Stimulation Of

ABSTRACT Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDH with DNA during unwinding, we examined the binding of TAgDH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDH requires ≥40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5′ junction on each strand, with 5 bp of duplex DNA and ∼17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5′ direction into the duplex region, while resulting in no significant changes to the 3′ edge of strongest protection. Our data indicate that each TAgH encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgH interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.

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Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

10.1101/811141 ◽

2019 ◽

Author(s):

Sujan Devbhandari ◽

Dirk Remus

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Point Mutations ◽

Dna Helicase ◽

Dna Unwinding ◽

Replication Forks ◽

Chromosomal Dna ◽

Replication Checkpoint ◽

Polymerase Domain ◽

Tightly Coupled

ABSTRACTThe coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Using the reconstituted budding yeast DNA replication system, we demonstrate that Pol ε variants harboring catalytic point mutations in the Pol2 polymerase domain, contrary to Pol2 polymerase domain deletions, inhibit DNA synthesis at replication forks by displacing Pol δ from PCNA/primer-template junctions, causing excessive DNA unwinding by the replicative DNA helicase, CMG, uncoupled from DNA synthesis. Mutations that suppress the inhibition of Pol δ by Pol ε restore viability in Pol2 polymerase point mutant cells. We also observe uninterrupted DNA unwinding at replication forks upon dNTP depletion or chemical inhibition of DNA polymerases, demonstrating that leading strand synthesis is not tightly coupled to DNA unwinding by CMG. Importantly, the Rad53 kinase controls excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint.

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p53 suppresses mutagenic RAD52 and POLθ pathways by orchestrating DNA replication restart homeostasis

10.1101/204057 ◽

2017 ◽

Cited By ~ 1

Author(s):

Sunetra Roy ◽

Karl-Heinz Tomaszowski ◽

Jessica Luzwick ◽

Soyoung Park ◽

Jun Li ◽

...

Keyword(s):

Dna Replication ◽

Genomic Instability ◽

Genome Instability ◽

Single Cells ◽

Replication Forks ◽

Mutational Signatures ◽

Development Of Resistance ◽

Cycle Arrest ◽

Replication Restart ◽

Dna Replication Restart

ABSTRACTClassically, p53 tumor-suppressor acts in transcription, apoptosis, and cell-cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks driving genomic instability independent of transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POLθ pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53 suppression of replication genome instability.

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Reconstitution of translesion synthesis reveals a mechanism of eukaryotic DNA replication restart

Nature Structural & Molecular Biology ◽

10.1038/s41594-020-0418-4 ◽

2020 ◽

Vol 27 (5) ◽

pp. 450-460 ◽

Cited By ~ 11

Author(s):

Thomas A. Guilliam ◽

Joseph T. P. Yeeles

Keyword(s):

Dna Replication ◽

Translesion Synthesis ◽

Replication Restart ◽

Eukaryotic Dna ◽

Dna Replication Restart

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DNA Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

Journal of Biological Chemistry ◽

10.1074/jbc.m407772200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45586-45593 ◽

Cited By ~ 30

Author(s):

David Shechter ◽

Carol Y. Ying ◽

Jean Gautier

Keyword(s):

Dna Replication ◽

Neutralizing Antibodies ◽

Atp Hydrolysis ◽

Initial Period ◽

Dna Helicase ◽

Dna Unwinding ◽

Origin Firing ◽

Replicative Helicase ◽

Mcm Proteins

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activitiesin vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) fromXenopus laeviseggs and six purified polyclonal antibodies generated against each of theXenopusMcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2–7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21cip1specifically blocks origin firing but does not prevent helicase action. When p21cip1is added, followed by the non-hydrolyzable analog ATPγS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

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Single-molecule binding characterization of primosomal protein PriA involved in replication restart

Physical Chemistry Chemical Physics ◽

10.1039/d1cp00638j ◽

2021 ◽

Author(s):

Tzu-Yu Lee ◽

Yi-Ching Li ◽

Min-Guan Lin ◽

Chwan-Deng Hsiao ◽

Hung-Wen Li

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Single Molecule ◽

Replication Forks ◽

Bacterial Survival ◽

Dna Damages ◽

Replication Restart

DNA damages lead to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal...

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